Hydroxy analog of vitamin b12 and processes for preparing the same



March 13, 1956 E. A. KACZKA ETAL HYDROXY ANALOG OF VITAMIN Bm AND PROCESSES FOR PREPARING THE SAME Filed July 20, 1950 u. o E 2 3 m m 5 w, n: I 2 E o O D .O m 2 I J In a. o

3 8 F i a 8 v m I a 1 o l 2 m o m I E z: 25

8 1;: o 3 Q n o D N I l 2 o O o o o u I) a 2 9 a If) N 1 2 INVENTORS EDWARD A. KAOZKA BY DONALD E.VIOLF KARL FOLKERS ATTORNEY addition to the cya" United States Patent -O 2 ,3 .nYnRoxY A GDFVITA I 1:11 M) PROCESSES FOR PREPARING THE SAME Edward A. Kaczka,ElizabethrbonaldE; WolfJ'i-inceton, V and Karl-Folkers,l'lainfielm' hl. J.,assignors to Merck V Ath -9 95 9; .59. Se l N new s (ilaims. cr m -s1 Inc.,' R"ahway, ,L, a corporation of New 2,738,301 Petented Mar. 13, 1956 0H- 11.0 Co on OH- x o X3170 It should, of course, be understood that these theoreticalexplanationsfor the possible structure of vitamins Blfifllld 'B z 'are based on our present knowledge of these products and does not exclude. the possibility that subsequent experirnental data. will be establish that the postulated structures are, in fact, incorrect. Accordingly, we do not wish to be bound by these theoretical considerations however l ikelyihey may appear to be in the light ience, our novel chemical substance has been designated vitamin 1312a,.

Vitamin B12 has been described by R i ckes at al,., in Science 107, 396-397 (11 948). in view "of the outstand: ing importance of vitamin B12. in the treatment of pernicious anemia, great interest has been centered in studying the constitution of this most potent compound in an effort to prepare it by eornplete synthesis, and to prepare medications thereof which might extend the usefulness of vitamin B12. p v

Unfortunately, in contrast to the other known vitamins, vitartiin B12 is found to be an exceedingly complex com pound having molecular weight in excess of 1300. Consequeutly, thus far ithas notibeen possible to determine the complete compositionof=vitamin B12, although some information of significant importance has resulted from" studies of the constitutionbf thisyitarnin. It hasbeen found a the qqmaauhd contain vcq alt, n f rther th t hd se aih sq d tio t ihhes iahd other s, it is belieyed that yitaminQBizis' a'coballt comp hnds. s h l c r ai o e 90 fli g ohPSLi va raviq y-Th structureof this compeuhd may be rep "esented'a's fol- W e, I e

rslh h c ba t e aai t h itiesed t th grou coordinated to the cobalt are also probably linked to each otherin' some'for'm as yet'uiikiiown'fi Vitamin B1241, the compound of our invention, maybe summer-ea to be the hydrbxy analog of'vitainin B12 whereiifa hydroxygroup has replaced the cyanidegroupin the o t yp hoy ii s Wer s b li wit of our present knowledge. These explanations are presented principally as a means for .proyiding a better understanding of our invention.

Vitamin B12; has important advantages over vitamin Biz. First, it is more readily obtained insome instances in pure fo'rm'than is yitamin B12. For example, vitamin B g; may be recovered relatively easily in pure form by crystallization. fromcertain solvent mixtures; whereas it'is sometimes diificult to obtain vitamin B12 in pure crystalline form from concentrates.

In preparing our novel chemical compound, we may utilize as the starting material, vitamin B12, which inits pure form is a red colored crystalline solid having refractive indices a, 1.619; 13,1649, 7, 1.659 after drying-at .C. in vacuo'until the indices are constant, and exhibiting strong absorption bands at 2780 A., 3610 A. and 5500 A. Vitamin B12 is described in more detail by Edward Riches et al. in Science, volume 107, pages 396 and 397 (1948).. Crystalline vitamin B12 is found to have an activity ,of about 11,000,000 units/mg. as determined by the 23-hour LLD factor assay of Shorb, J.

Biol. Chem., 1946, 163, 393. Various methods suitable for preparing vitamin B z are described in UnitedStates patent application Serial No. 20,'35,6, filed April 10, 1948,

- now abandoned.

Alternatively, we have found that vitamin B122. canbe isolated-from concentrates ofyitamin B zobtained from fermentetion sources. :[hes e crude concentrates of vitaminBiz contain substantial amounts of vitamin B12& which can be isolated by suitable process such as fractional crystallization from solvents.

In accordance with one embodiment of our invention vitamin B12 .orzcondentrates-thereof are reacted with hydrogen and acatalyst and vitamin B128, is recovered.

A crude vitamin B12 eoncentrate or a purified form of vitamin B12 is dissolved in water, an alcohol or aqueous mixture thereof. The solution containing vitamin B12 thus formed is reacted with hydrogen in the presence .of a hydrogenation catalyst such as Raney nickel, the noble metals or the noble metal oxides. The reaction is conveniently carried out at approximately room temperature and at substantially atmospheric pressure. After about three mols of'hydrogen per mol of vitamin B12 has been absorbed, the rate of hydrogen consumption decreases appreciably, and the removal of the cyanide radical from vitamin B1 is substantially complete. During the reduction, the colorof thereaction mixture changes from the characteristic red color of vitamin B12 to a brown color.' It is preferable to stop the reaction after the required three mols of hydrogen have been absorbed, since further reduction may result in more extensive changes.

When the reduction is complete, the suspended catalyst is removed from the solution which on rcoxidation by exposure to air changes in color from brown to red. By evaporating the resulting red solution, vitamin B12a1$ obt'aine'din solid form. The vitamin BlZa is prepared in crystalline form by dissolving the residue so obtained in a small amount of water, and adding acetone to the resulting aqueous solution. Upon allowing the aqueousacetoiiesolution to stand at room temperature, the vitamin B1211 crystallizes and may be separated from the mother liquors by filtration or centrifugation. Alternatively the aqueous solution obtainedafter removing the suspended hydrogenation catalyst may be diluted with about 7 or 8 volumes of acetone, and the resulting aqueous-acetone solution allowed to stand until crystallization of the vitamin B123. is complete.

In accordance with a further embodiment of our invention, we find that vitamin 1312;; can be recovered from fermentation broths produced by the propagation of various microorganisms such as certain strains of S. griseus, 'S. aureofaciens, and the like For example, when a streptomycin or grisein-producing.strain of S. griseus is propagated in a medium which contains a source of cobalt and is suitable for the production of streptomycin or grisein, a concentrate can be recovered from the resulting broth containing vitamin B12, vitamin B1211, and associated impurities Such a concentrate is obtained for example, by treating a broth with activated charcoal to effect adsorption of the active substances, eluting the charcoal with aqueous pyridine. concentrating the eluate to dryness, dissolving the residue in alcohol, chromatographing on activated alumina, and crystallizing the active fractions from water. The vitamin B12 in such a concentrate can be separated from the vitamin B123. by extracting an aqueous solution of the concentrate with portions of benzyl alcohol until no more vitamin B12 is removed. It is possible to remove vitamin B12 by this method since vitamin B12 has a water/benzyl alcohol distribution coefficient of about 1.2 while the distribution coeflicient of vitamin B1211 in these solvents is about 8.' After separating the benzyl alcohol layer from the water layer, the vitamin B123 can be recovered by concentrating the water mixture and recrystallizing the residue from water by the addition of acetone. Alternatively, other methods such as fractional crystallization of the crude concentrate from a water-acetone mixture can be utilized to recover vitamin B1211 in crystalline form directly. The vitamin B1211 can be readily identified by means of its ultraviolet absorption spectrum as is more completely described hereinafter.

An aqueous solution of crystalline vitamin B122 prepared by any of the previous methods exhibits the following principal absorption maxima:

in A. 1cm.

Shoulder or Broad Band. Strong Band Band 2,700 to 2,770 3,520 to 3,540 5,200 to 5,300

In addition there is a faint band at 4150 A. with is the optical density of a 1% solution of the substance in a cell 1 cm. in length as determined at the particular light wave length. While the curves are somewhat similar in general appearance, it will be seen that absorption maxima of the two substances are distinctly different. Accordingly the ultraviolet spectrum in conjunction with the other properties serve to distinguish vitamin 13m from vitamin B12.

Vitamin B123 is a compound having the properties of a weak base which can be titrated with acid. Since it readily forms salts, it is important in the process of preparing vitamin B129, that it be kept free from acids, acidforming substances, or anions other than hydroxyl. in its chemical reactions, vitamin B1211 can be considered to be the hydroxy analog of vitamin B12, i. e., it may be thought of as a compoundwherein a hydroxy group has replaced the cyanide group of vitamin B12.

Vitamin B1211 has been found to be capable of promoting the growth of the microorganisms, Lactobacillus lacti's Dorner and L. leichmannii, and therefore these organisms may be used as a means of assaying this product. In the following table are shown typical assay results of samples of vitamin B123. obtained by hydrogenating vitamin B12, and from S! griseus, in comparison with vitamin B12.

It will be noted from the foregoing data that the micro biological assays of a sample of vitamin B123, prepared by hydrogenating vitamin B12 and a sample of the same product obtained from S. griseus are about the same Further, it will be seen that although by the titrimetric method of assay with L. lactis vitamin B128. appears to be only one half as active as vitamin B12, by the cup assay with this same microorganism it is fully as active as vitamin B12. In the L. leichmanm'i assay, vitamin B12 and vitamin B121]. are equally active if added aseptically to sterile culture medium. However, if autoclaved with the medium vitamin 1312s is inactivated and shows only 17% as much activity as vitamin B12. The cup assay procedure is described in Science 110, 507 (1949), the L. lactz's tube (titrimetric) procedure in J. Biol. Chem. 180, 125 (1949), and the L. leichmarmii tube procedure in J. Biol. Chem. 175, 475 (1948).

The red crystals of vitamin B1211 are needle-like or blades whose crystal system is orthorhombic. The indices of refraction for the crystalline product dried at room temperature in vacuo are about a, 1.580; 5, 1.640; and 7, 1.656. Heating the product in vacuum at 100 C. does not alter the 13 and 'y indices but causes a progressive increase in the or index. Thus, on heating a sample of vitamin B122 at 100 C. for two hours in vacuo, the refractive indices were found to be about a, 1.604; p, 1.640; and 7, 1.654.

Vitamin B1211 is relatively soluble in water, lower aliphatic alcohols and aqueous alcohol mixtures. It is relatively insoluble in solvents such as ether, acetone, chloroform, carbon tetrachloride, benzene, toluene, petroleum ether, and the like. Vitamin B123. is soluble to the extent of about 3.6 mg. per ml. in a mixture containing acetone and 20% water (by volume) and to the extent of Table II.

area

min Bias is converted to vitamin 1343,, Analogs may also be prepared by reactingvitainin' Bis-with a salt of the anion corresponding to the analog f-fondxamplc, by reacting vitamin 3,120. with an alkali metal nitrite, thiocyanate, or iodide it isjconvertd to the nitrite, thiocyanate or iodide analog respectively. T i

The followingare appgqximateanalyses of two samples of am n .1 s obta ed n q p da ss vani a-pre o y described processes 1' Nitrogen, P o p o s-y."- ti. 19$ de ermined Cobalt 416, t ptdstsnainstl) C r n hydrogen; nd are a lts a e Vitamin B129, gradually darkens with charting and decomposition and without meltingon heating. Depending upon the rate of heating, it darkens at ahout 200 0., and does not melt on heating to- 300 C.

' During the reduction-of vitamin Biz-to vitamin B125 the color of the solution changes from the characteristic red color of vitamin Biato a brown color; This brown solution on exposure 'to airt-urns to a deep red icolor characteristic of vitamin 312a. Thisred color is differentthan that of'solutions of vitaminBm.

For comparison and summary, "the eriteria'which serve to differentiate vitamin Biz and vitamin Bim' are listed in "Ti tle 11 Ta l ill--Ast i iv tq ir Pre ace Wt. Incre- Dose Fed No. of

substance Daily, g. Male Rats fg g f Control (undosed) 10 27 Vitamin B1: 0.063 10 56 Vitamin B12... 0.125 10 61 Vitamin 1312-. 0.063 10 Vitamin B1 0.125 N 10 59 "Vitamin B129. has also been tested clinically and has been. fou d o, be etfeet re in promotin cl ni al improve- ,ment and in producing positive hemopoietic responses in patients with Addison's pernicious anemia, tropicalsprue,

non-tropical sprue, and nutritional macrocytic anemia.

Because of extreme variability from patient to patient, comparative studies on the effect per unit of weight of vitamin- B12 and B125 will. take ..some time to evaluate. However, it hadbeen established that vitamin B12a, like vitamin Brz, produces a rise in reticulocy tes, red. blood cells, white blood cells, platelets and'hemoglobin, and promotes a return of the bone marrow to normal.

I The preparation of our novel compound, vitamin Bu possessing pronounced therapeutic value in the treatment of pernicious anemia, and possessingproperties which r nalce possible the convenient recovery of the compound in pure form, may be illustrated by the following specific examples.

Exarriple 1 solution containing 25.3. mg. of vitamin B12 in. 15

nrrl. offwater was shaken with 78, mg. of platinum oxide catalyst and hydrogen gas under substantially atmospheric pressure at 25 C. for 20 hours. iHydrogen was absorbed.

paring the absorption ofihydrogen the color of the solution changed from redto. brown. The solution was separated from the catalyst and evaporated to dryness in vacuo. The residue was then dissolved in 1 ml. of water and then diluted with about 6 ml. of acetone. After stand- Property Vitamin B Refractive Indices i (11.580 (after drying at room temperature in vacuo).

Absorption spectra in water Shoulder or broad band 2,700-2,770A., 137'. Strong maximum 3,5203,540A:, 150; 7 Maximum 5,200-5,30 0A., 58. V

Distribution coetlieient for benzyl alcohol-water i 215 0 6 J .anen's, Meltmg point Does not melt up to 300 C Does not melt up to 360 C.

Five waves of which only first is resolved. 1.0I1ic=0.34 microampsJmgJec. and "E% v. 2-5. combined give E=0.2 to 1.2 v. and

Activity for growth of LLD r .i 11,00dunits/rnicrogrnrn Q V unitS/micmgmm 11,000 unitslmicrogram (cup assay). about 8.0.

Darkens at about 200 C.

1 All run in 0.1 N lithium borate b tter, pH 9, at as that ia wi gl 2/; 5% i 1 Concentration in'wa'tei. eoneentration' in solvent.

.mili B12.

ing for several hours a small amount of precipitate (about 2-3 mg.) was formed and was then separated from the solution. This solution was diluted with an additional 2 ml. of acetone and again allowed to stand for several hours. During this time about 4-5 mg. of noncrystalline precipitate formed. This solid was separated from the solution and an additional 2 ml. of acetone was added to-the solution. On standing vitamin Bizs began to crystallize inthe form of red needles. After standing for '24 shouts, the'crystalline material was separated, yield V 1 cm.

)\ in A. 15

Shoulder or Broad Band 2, 750 128 Strong Band 3, 525 154 Band 5, 300 56 Example 2 A solution of 16.5 mg. of vitamin B12 in 12 ml. of water was shaked with 48 mg. of palladium oxide catalyst and hydrogen gas under substantially atmospheric pressure at 25 C. for 30 hours. On absorption of hydrogen the solution turned from red, to brown in color. After no further hydrogen was absorbed the solution was filtered and evaporated to dryness in vacuo'. The microbiological assay showed the residual material to have an LLD activity value of 3000 to 5000 units per microgram.

Example 3 Nineteen and two-tenths mg. of a crude concentrate of vitamin B12 was treated in water solution with hydrogen and a palladium catalyst at 25 C. After absorption of hydrogen was complete, the catalyst was removed leaving a yellow-colored solution. Microbiological assay of the residue showed the material to have an LLD activity of 96 units per microgram. This low LLD value is attributed to the fact that a crude starting material con taining impurities was used.

Example 4 Twenty-six mg. of vitamin B12 was dissolved in 14 ml. of water and then shaken with about 100 mg. of Raney nickel catalyst under a slight hydrogen pressure for two hours at 25-26" C. The dark-colored solution was separated from the catalyst by centrifugation and then concentrated to a volume of 6-8 ml. in vacuo to about 28 C. The concentrated solution was diluted with about volumes of acetone and then allowed to stand at 26-28 C. After standing for several hours dark red needle-like crystals of vitamin B123. separated from solution. The first crop of crystals weighed 5.2 mg. and the absorption spectrum of an aqueous solution showed maxima at 2750 A., 3525 A., and 5300 A.

After recrystallization the crystals exhibited the typical absorption spectrum of vitamin B12 shown on page 6.

Example 5.--Vitamin Biza Twenty-eight and three-tenths milligrams of vitamin B12 was dissolved in 14 ml. of water containing 93 mg. of previously reduced platinum oxide catalyst. This solution was stirred under a slight hydrogen pressure for seventeen and one-quarter hours. During the absorption of hydrogen the color of the solution changed from red to brown. On subsequent exposure to air, the resulting solution slowly turned red. The catalyst was separated from the solution by centrifuging and then the solution was concentrated to a volume of 7-8 ml. This solution was diluted with about 7-8 volumes of acetone and crystallization began after standing at room temperature for a short time. Yield of vitamin 8129., 16 mg.

The above experiment was repeated using 107.3 mg. of vitamin B12. The yield of vitamin B128. after one recrystallization from acetone-water was 59 mg. The refractive indices of these crystals of vitamin 1312s. were a, 1.580; 8, 1.640; 7, 1.656 after drying at room temperature in vacuo, and a, 1.604; 5, 1.640; 7, 1.654 after drying at C. in vacuo.

The crystalline vitamin Bm from the above experiments was combined and recrystallized from water by the addition of acetone. Yield 56 mg. These crystals were shown to be at least 98% pure by solubility analysis.

' The absorption spectrum in aqueous solution at natural pH sho'wed principal maxima at about 7 3525 A. an... a faint band at about 4150 A. (Elfi 29) and a broad band at 2700-2770 A. (Elg 137) Analysis.Found: N, 13.75; P, 2.18; Co, 4.64.

Example 6 A streptomycin broth prepared by cultivating a streptomycin producing strain of S. griseus in a liquid nutrient medium containing soybean meal was worked up in the usual way to remove the mycelium and suspended solids. The filtered broth was then passed through a column containing a carboxylic acid resin, a granular copolymer of methacrylic acid and divinyl benzene containing 5% divinyl benzene, which removed the streptomycin.

The resulting spent broth was treated with activated charcoal to absorb the substances capable of promoting the growth of Lactobacillus lactis Dorner. The active substances were then eluted from the charcoal with an aqueous-pyridine or aqueous-picoline solution and concentrated to remove the pyridine or picoline. Methanol was added to the resulting solution causing the precipitation of impurities which were removed by filtration. Acetone was then added to the filtrate resulting in the precipitation of the active substances which were separated from the solution and extracted with methanol. The active substances were precipitated from the methanol by the addition of acetone, separated, and extracted again with methanol. This purification procedure was repeated several times, and then the active substances were precipitated from the methanol solution by adding ethyl ether. This precipitate was dissolved in methanol and chromatographed over activated alumina. The column was then developed with methanol to obtain a main redcolored band which was finally eluted with methanol. Ether was then added to the rich methanol eluate precipitating the active material which was separated and extracted with methanol. This chromatographic purification procedure was repeated twice more, and the active substances finally precipitated from the rich eluate with ether. The product so obtained was separated and dissolved in water. Upon the addition of acetone to this aqueous solution a crystalline product containing the active substances precipitated and was recovered.

Fifteen and seven-tenths milligrams of this red crystalline mixture which was shown to contain about 25% of vitamin B12, was dissolved in 3 ml. of water and extracted seven times with 2 to 3 ml. portions of benzyl alcohol. The aqueous solution was then extracted several times with ether to remove dissolved benzyl alcohol and then traces of ether removed in vacuo. The aqueous solution was then diluted with about 5-7 volumes of acetone and after standing for a short time, crystallization began. The dark-red needles that formed were separated from the solution, washed with acetone and dried. Yield, 6 mg. of vitamin B123. The absorption spectrum in aqueous solution at the natural pH showed principal maxima at about 2740-2750 A., 3530-3540 A. and 5200-5300 A. characteristic of vitamin B1211.

9 tallineproduct from. culture-,: filtrates of, Sp igi isegs, prepared as described in Example 6 W385 dijssolveddrulQ-ml. of water and this solution WaS,dl. d6d} into, two, equal parts. partand the solutions allowedto stand at about 25-30? C.

and after a short time a precipitate formed in eachs lu-' talline precipitate E. The mother liquor of precipitate E was diluted to a total volume. of 120 ml. with acetone. This solution after standing for'a short time gave a crystalline precipitate P which after separation, washing with acetone and drying weighed 22.4 mg. This compound was fractionally crystallized'from water by dilution with acetone. The first two fractions which were collected weighed ca. 6.5 mg. The third fraction was used for comparison with vitamin B1221, obtained from vitamin B12 by reaction with hydrogen in presence of a catalyst. The refractive indices of the crystalline product dried at ca. 25 C. were a, 1.584; [3, 1.640; 1.657 which after drying at 100 C. two hours in vacuo changed to a, 1.608; ,6, 1.640; 7, 1.656.

Example 8 Three hundred and fifty milligrams of vitamin B12 was dissolved in 75 ml. of water containing 300 mg. of reduced platinum oxide catalyst. This solution was shaken. under a slight hydrogen pressure for forty minutes. During the absorption of hydrogen the color of the solution changed from red to brown. On subsequent exposure to air the solution slowly turned red. This solution containing suspended catalyst was diluted with ca. 400 ml. of acetone. After allowing the solution to stand at room temperature for a short time, the dispersed catalyst coagulated and was easily removed by centrifugation. After removal of the catalyst, the solution was diluted to a volume of ca. 900 ml. with acetone. The solution was allowed to stand overnight during which time crystallization took place in the form of dark red needles. The first crop of crystals weighed 200 mg. After one recrystallization from water by dilution with acetone, vitamin B1221 of 98 per cent purity by solubility analysis was obtained.

AnaL-Found: N, 13.33.

The ultra-violet absorption spectrum in water at natu ral pH showed maxima at about 2700-2770 A., 3520- 3530 A., and 5200-5300 A.

It should be understood that various changes may be made in our process as herein described without affecting the improved results attained. Thus various modifications in conditions as to time, temperature, etc., and various changes in procedure differing from those herein given as illustrative of the preferred embodiments of our invention may be made without departure from the scope thereof. Accordingly, the scope of our invention is to be determined in accordance with the prior art and appended claims.

We claim: 1

1. In the process for converting vitamin B12 to its hydroxy analog wherein the cyanide group of the vitamin B12 molecule is replaced by a hydroxy group, herein termed vitamin Blfla, the step which comprises reacting vitamin B1 with hydrogen in the presence of a hydrogenation catalyst.

2. The process for converting vitamin B12 into its hydroxy analog, herein termed vitamin B12, that com- Twenty-five ml. of acetone was, addedto each,

#10 prises actin nimus ol t nfi tam n-B12 having a characteristic red, color with hydrogenjnthe presence of a hydrogenation catalyst, continuing the reaction until hydrogenation is complete as evidenced by a changein color of the solution fromred to brown, removing the catalyst, and exposing-the, solution: tooxygen containing gas untilthe brown color changes back to red.

3. The process as definedin claim 2'wherein the hyforming salts with acids, containing the elements carbon,

hydrogen, nitrogen, phosphorus, cobalt and oxygen, further characterized by having ultraviolet absorption maxima at about 270-277 m 352-354 III/1., 520-530 III/1., being soluble in water, lower aliphatic alcohols and aqueous alcohol mixtures, being insoluble in ether, acetone, chloroform, carbon tetrachloride, benzene, and toluene, forming red crystals having indices of refraction of about a, 1.580; 5, 1.640; and 7, 1.656; after drying at 25 C. in vacuo by being convertible into vitamin E12 by reaction with solutions containing cyanide ions, and by having an LLD activity of at least 6,600,000 LLD units per milligram.

5. The process for separating the hydroxy analog of vitamin B12 wherein the cyanide group of the vitamin B12 molecule is replaced by a hydroxy group, herein termed vitamin 312a from a concentrate containing vitamin B122 and vitamin B12 obtained from a fermentation broth which comprises extracting an aqueous solution of said concentratewith benzyl alcohol to remove the vitamin B12, and recovering ,vitamin 1312s from the resulting aqueous solution. Y

6. The process forrecovering the'hydroxy analog of vitamin B12 wherein the cyanide group of the vitamin B12 molecule is replaced by a hydroxy group, herein termed vitamin B129. from a concentrate containing vitamin B123.

and vitamin B12 obtained from a streptomycin fermentation broth which comprises extracting an aqueous solution of said concentrate with benzyl alcohol to remove sub- 1 crystalline vitamin B1221.

7. The process for recovering the hydroxy analog of vitamin B12 wherein the cyanide group of the vitamin B12 molecule is replaced by a hydroxy group, herein termed vitamin B1221. from a concentrate containing vitamin B12. and vitamin B12 which comprises, adding acetone to an aqueous solution of said concentrate causing a crystalline precipitate to form, separating said precipitate, adding additional acetone to the mother liquors from said precipitate causing the precipitation of a second crystalline product, and repeating said fractional crystallization procedure to recover a crystalline fraction consisting of substantially pure vitamin 1312a.

8. The hydroxy analog of vitamin B12 wherein the cyanide group of the vitamin B12 molecule is replaced by a hydroxy group which is a red crystalline substance effective in the treatment of anemias substantially free from cyanide groups containing the elements carbon, hydrogen, nitrogen, phosphorus, cobalt and oxygen, further characterized by having ultraviolet absorption maxima at about 273 m 351 m 525 m being soluble in Water, methanol and ethanol, being insoluble in ether, acetone, chloroform and toluene, being convertible into vitamin B12 by reaction with solutions containing cyanide ions, and by having an LLD activity of at least 6,600,000 units per milligram.

(References on following page) References Cited in the file of this patent Kaczka: Jour. of the Amer. Chemical Society, vol. 71,

/ Apr. 1949, pp- 1514, 1515.

UNIT?!) STATES PATENTS Smith: Nature, Apr. 24, 1948, vol. 161, pp, 638, 639. 137L775 Major 1932 Smith: Proceedings of the Biochemical Society in the 2563794 Rlckes 1951 5 Biochemical Journal, vol. 43, No. 1, Oct. 1948, p. VIII,

OTHER REFERENCES R' k S D 3 1948 634 635 Dounce: Science, vol. 97, Jan. 1, 1943, pp. 21, 22, 23. E meme Spies: Southern Med. Jour.,Jan. 1950, p. bu. Somme 1948 Zuckcr: Vitamins and Hormones, vol. 8, (1950) p. 7.

Girdwood: Lancet, Aug. 20, 1949, p. 346. 

8. THE HYDROXY ANALOG OF VITAMIN B12 WHEREIN THE CYANIDE GROUP OF THE VITAMIN B12 MOLECULE IS REPLACED BY A HYDROXY GROUP WHICH IS A RED CRYSTALLINE SUBSTANCE EFFECTIVE IN THE TREATMENT OF ANEMIAS SUBSTANTIALLY FREE FROM CYANIDE GROUPS CONTAINING THE ELEMENTS CARBON, HYDROGEN, NITROGEN, PHOSPHOROUS, COBALT AND OXYGEN, FURTHER CHARACTERIZED BY HAVING ULTRAVIOLET ABSORPTION MAXIMA AT ABOUT 273 MU, 351 MU, 525 MU, BEING SOLUBLE IN WATER, METHANOL AND ETHANOL, BEING INSOLUBLE IN ETHER, ACETONE, CHLOROFORM AND TOLUENE, BEING CONVERTIBLE INTO VITAMIN B12 BY REACTION WITH SOLUTIONS CONTAINING CYANIDE IONS, AND BY HAVING AN LLD ACTIVITY OF AT LEAST 6,600,000 UNITS PER MILLIGRAM. 